ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■
Contents lists available at ScienceDirect
Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
SKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma Laura Evans a, Lindi Chen a, Giorgio Milazzo b, Samuele Gherardi b,c, Giovanni Perini b,c, Elaine Willmore a, David R. Newell a, Deborah A. Tweddle a,* a
Newcastle Cancer Centre at the Northern Institute for Cancer Research, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK Department of Pharmacy and Biotechnology, University of Bologna, Via F. Selmi 3, Bologna 40126, Italy c Health Science and Technologies-Interdepartmental Centre for Industrial Research (HST-ICIR), University of Bologna, Via Tolara di Sopra 41/E, Ozzano Emilia (Bologna) 40064, Italy b
A R T I C L E
I N F O
Article history: Received 20 January 2015 Received in revised form 26 March 2015 Accepted 28 March 2015 Keywords: SKP2 MYCN Neuroblastoma G1 arrest Apoptosis
A B S T R A C T
SKP2 is the substrate recognition subunit of the ubiquitin ligase complex which targets p27KIP1 for degradation. Induced at the G1/S transit of the cell cycle, SKP2 is frequently overexpressed in human cancers and contributes to malignancy. We previously identified SKP2 as a possible MYCN target gene and hence hypothesise that SKP2 is a potential therapeutic target in MYCN amplified disease. A positive correlation was identified between MYCN activity and SKP2 mRNA expression in Tet21N MYCN-regulatable cells and a panel of MYCN amplified and non-amplified neuroblastoma cell lines. In chromatin immunoprecipitation and reporter gene assays, MYCN bound directly to E-boxes within the SKP2 promoter and induced transcriptional activity which was decreased by the removal of MYCN and E-box mutation. Although SKP2 knockdown inhibited cell growth in both MYCN amplified and non-amplified cells, cell cycle arrest and apoptosis were induced only in non-MYCN amplified neuroblastoma cells. In conclusion these data identify SKP2 as a direct transcriptional target of MYCN and supports SKP2 as a potential therapeutic target in neuroblastoma. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction MYCN amplification is found in 25% of neuroblastoma and is strongly associated with advanced-stage and aggressive disease [1,2]. Although different strategies for inhibiting MYCN have been investigated, direct therapeutic targeting of the oncoprotein presents a significant challenge [3,4]. We have previously identified a number of genes involved in the regulation of the G1 checkpoint whose expression changes in relation to MYCN status [5]. One potential MYCN target gene is the F-box protein S-phase kinase-associated protein 2 (SKP2) which is the substrate recognition subunit for the SCFSKP2 E3 ligase complex. Implicated primarily in G1/S progression, SKP2 targets cell cycle regulators (e.g. p27KIP1, p21CIP1 and p57KIP2) for ubiquitination and degradation, thereby promoting cell cycle progression. Overexpressed in multiple cancer types, the main oncogenic contribution of SKP2 is attributed to targeting the CDK inhibitor
Abbreviations and acronyms: SKP2, S-phase kinase-associated protein 2; SCF, SkpCullin-F-box containing complex; ChIP, chromatin immunoprecipitation; CDK, cyclin dependent kinase; MNA, MYCN amplified; NCS, negative control siRNA. * Corresponding author. Tel.: +44 0 191 208 4421; fax: +44 (0) 191 208 4301. E-mail address: [email protected] (D.A. Tweddle).
p27KIP1 (p27 hereafter) for degradation, with the inverse relationship of high SKP2 and low p27 level often correlating with poor patient prognosis [6–8]. SKP2 overexpression has been identified as an indicator of highrisk neuroblastoma and found to correlate with low p27 protein levels and high E2F activity, suggesting SKP2 contributes to disease development and progression [9]. Moreover, RNA studies have previously demonstrated a relationship between MYCN and SKP2 expression in primary neuroblastoma tumours and cell lines [5,9], suggesting SKP2 may be a potential MYCN target gene. SKP2 has recently been identified as a direct c-MYC target gene [10]. Due to the homology within the Myc family and functional overlap often observed between c-MYC and MYCN, established c-MYC targets are often automatically presumed to be regulated by MYCN. However, different expression patterns are observed for these transcription factors in normal development and as oncogenes they are involved in distinct tumour types (e.g. c-MYC in Burkitt’s lymphoma and MYCN in neuroblastoma). Furthermore not all c-MYC gene targets are found to be expressed in MYCN-expressing neuroblastoma cells most probably due to the reported differences in E-box selection between MYCN and c-MYC [11], as reviewed in Refs. 12 and 13. We therefore hypothesise that SKP2 is a direct MYCN target gene and contributes to the aggressive phenotype of MYCNamplified tumours.
http://dx.doi.org/10.1016/j.canlet.2015.03.044 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Laura Evans, Lindi Chen, Giorgio Milazzo, Samuele Gherardi, Giovanni Perini, Elaine Willmore, David R. Newell, Deborah A. Tweddle, SKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.03.044
ARTICLE IN PRESS L. Evans et al./Cancer Letters ■■ (2015) ■■–■■
2
To investigate this hypothesis, we assessed the correlation between MYCN and SKP2 protein, and SKP2 mRNA expression in the conditional MYCN-expressing SHEP Tet21N cells and a panel of MYCN amplified and non-amplified neuroblastoma cell lines. Using chromatin immunoprecipitation (ChIP) and reporter gene assays, this is the first study to show that SKP2 is a direct MYCN transcriptional target. In addition, using SKP2 siRNA knockdown we highlight the therapeutic potential of targeting SKP2 in neuroblastoma.
Chromatin immunoprecipitation (ChIP assay) The ChIP assay was performed as previously described [22], and the antibodies employed were: MYCN (B8.4.B, C-22, Santa Cruz,), c-MYC (N-262, Santa Cruz) and MAX (C-17, Santa Cruz). Samples were analysed using SYBR Green quantitative PCR with the specific primers listed in the Supplementary material (Table S1). The known MYCN target gene ornithine decarboxylase (ODC1) was used as a positive control using forward primer 5′- ATCACTTCCAGGTCCCTTGC-3′ and reverse primer 5′-GAGAGCGGAAAAGGGAAATC-3′. Luciferase assay
Materials and methods Cell culture and reagents MYCN-amplified neuroblastoma cell lines used were LAN5 [14], IMR32 [15] and p53 mutant IMR-KAT100 [16] and SK-N-BE(2c) [17] cells. Non-MYCN amplified cell lines used were SHSY5Y [17], GIMEN [18] and p53 mutant SKNAS [19]. Tet21N cells were cultured in media containing 1 μg/ml tetracycline (Sigma-Aldrich) for 24 hrs prior to experiments to switch off MYCN expression [20]. The identity of all cell lines was confirmed by karyotyping and was consistent with published reports. Cells were cultured in RPMI medium (Sigma) with 10% (v/v) foetal bovine serum (FBS). A total of 200 μg/ml of G418 antibiotic (Sigma-Aldrich) was added to the Tet21N cell medium. All cell lines were tested regularly and found to be free from Mycoplasma.
Quantitative reverse transcription PCR (qRT-PCR) qRT-PCR was performed as previously described and analysed using the ΔCT/ ΔΔCT method [5]. All primers and probes were inventoried using TaqMan Gene Expression Assays (Applied Biosystems) and mRNA expression data normalised to GAPDH.
The SKP2 reporter construct -1148-SKP2 and pGL3basic vector were kindly donated by Prof. Javier Lèon (University of Cantabria, Spain) [10]. pGL3-MutEB was generated by site-directed mutagenesis replacing identified E-Boxes (CACCTG) and (CCCGTG) with CTGCAG, and confirmed by sequencing. Cells were transfected with 0.8 μg of reporter constructs and 0.1 μg of β-galactosidase (pCMV.SPORT.βgal, Invitrogen) using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured using the luciferase assay system from Promega (E4030), corrected for nontransfected controls and normalised using β-gal activity to control for transfection efficiency. Statistical analyses All statistical tests were two sided and performed using GraphPad Prism (Graphpad Software, Inc.). The level of significance was taken to be p ≤ 0.05.
Results SKP2 protein expression correlates with MYCN expression in MYCNregulatable Tet21N cells
Western analysis Western blotting was performed using previously described methods [21]. Primary antibodies used were SKP2 (32–3300, Invitrogen) at 1:1000, MYCN (SC-53993, SantaCruz) at 1:500, p27 (SC-528, Santa-Cruz) and p21 (OP64, Calbiochem) both at 1:100 and GAPDH (SC-25778, Santa-Cruz) at 1:1000. Secondary goat anti-mouse/rabbit horseradish peroxide-conjugated antibodies (P0447/P0448, Dako) were used at 1:1000.
Cycloheximide treatment Tet21N cells were treated with final concentrations of tetracycline (1 μg/ml) and/ or 25 μmol/l cycloheximide (Calbiochem), alone or in combination. Cells were harvested at the indicated times and analysed using Western blotting.
RNA interference siRNA (40 nM) was transfected into cells using Lipofectamine 2000 reagent (Invitrogen) which was replaced with RPMI medium containing 10% (v/v) FBS after 24 hr. Three different oligonucleotides targeting SKP2 were synthesised by Eurogentec and the most effective duplex used in all knockdown experiments. siRNA targeting CDKN1B (p27) was verified and purchased from Qiagen (SI02621990). The sense strand of the SKP2 siRNA sequence chosen was: GUGAUAGUGUCAUGCUAAA. Inhibition of expression was confirmed by Western blotting against a negative control siRNA (NCS).
Cell proliferation assay Cell viability was measured by the sodium 3′-{1-(phenylaminocarbonyl)-3,4tetrazolim]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) colorimetric assay (Roche Applied Science) according to the manufacturer’s instructions.
Flow cytometry Cells were harvested, washed in cold PBS, fixed in 70% (v/v) ethanol and stored at 4 °C. Fixed cells were rinsed twice with PBS and treated with propidium iodide (PI) solution containing 40 μg/ml PI (Sigma) and 0.1 mg/ml RNase A (Sigma) at 37 °C for 30 minutes. Stained cells were analysed by a FACSCalibur cytometer (BD Bioscience, Oxford, UK).
Tet21N MYCN- cells showed an ~3-fold reduction in SKP2 mRNA 24 hr after tetracycline addition (Fig. 1A), which corresponded with decreased SKP2 protein expression over the 5 day tetracycline exposure period (Fig. 1B). Reduced SKP2 expression was associated with increased protein levels of the SKP2 targets p27 and p21CIP1 (p21 hereafter), suggesting that stabilisation of these CDK inhibitors induced the growth inhibition observed in the exponentially proliferating Tet21N MYCN− cells (Fig. 1C). To confirm that stabilisation of the CDK inhibitors was a result of decreased SKP2 protein expression, Tet21N MYCN+ cells were treated with a combination of tetracycline and the mRNA translation inhibitor cycloheximide (25 μmol/l, Fig. 1D). Cycloheximide treatment decreased MYCN and SKP2 protein expression (lanes 3 and 4), mirroring the effect of tetracycline only (lanes 5–7), and reduced the level of p27 and p21. Only p27 protein was still present, although at a reduced level, after combination treatment (lanes 8 and 9) suggesting that the loss of SKP2 in Tet21N MYCN− cells resulted in the post-translational stabilisation of p27, but not p21. Data analysis from the Versteeg-88 dataset of clinical neuroblastoma tumour samples by the R2 microarray analysis and visualisation platform (http://r2.amc.nl) [23] showed that expression of SKP2 and MYCN were strongly correlated (Supplementary material, Fig. S1 [23]) which was confirmed in two additional data sets; Maris-101, r = 0.520, p = 2.6e−8 and Jagannathan-100, r = 0.528, p = 1.7e−8 (figures not shown, http://r2.amc.nl). To further investigate the relationship between MYCN and SKP2, mRNA and protein levels were analysed in a panel of MYCN amplified (MNA) and nonMYCN amplified (non-MNA) neuroblastoma cell lines. MYCN protein levels correlated with SKP2 mRNA levels (Pearson’s correlation, r2 = 0.62, p < 0.05, Fig. 1E) but not SKP2 protein expression (Fig. 1F) suggesting that MYCN may influence the numerous mechanisms reported to regulate SKP2 protein stability.
Caspase-3/7 assay After 24 hr siRNA exposure a 1:1 volume of Caspase-Glo 3/7 reagent (Promega) was added to each well and incubated in the dark for 1 hr at room temperature. The plate was analysed on a microplate luminometer (Berthold Technologies, Hertfordshire, UK).
MYCN sensitises Tet21N cells to SKP2 knockdown SKP2 protein was successfully knocked down using siRNA (Fig. 2a), and resulted in a clear increase in p21 protein levels independent
Please cite this article in press as: Laura Evans, Lindi Chen, Giorgio Milazzo, Samuele Gherardi, Giovanni Perini, Elaine Willmore, David R. Newell, Deborah A. Tweddle, SKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.03.044
ARTICLE IN PRESS L. Evans et al./Cancer Letters ■■ (2015) ■■–■■
3
Fig. 1. SKP2 expression is associated with MYCN expression in neuroblastoma cell lines. (A) Normalised expression of SKP2 mRNA levels in Tet21N MYCN+ and MYCN− cells following 24 hr exposure to tetracycline (paired t test, p ≤ 0.05). (B) Western blot analysis of protein expression of SKP2, p27 and p21 in Tet21N cells in the presence and absence of tetracycline (C) Tet21N MYCN+ cells proliferate more rapidly than Tet21N MYCN− cells. (D) Western blot analysis of MYCN, SKP2, p27 and p21 protein levels in Tet21N cells following treatment with tetracycline (TET) and cycloheximide (CHX). MYCN protein acted as positive control due to its short half-life (~2 hr). (E and F) Relationship of MYCN and SKP2 at transcript (E) and protein (F) level in a panel of neuroblastoma cell lines (Spearman correlation, r = 0.62, p ≤ 0.05).
of MYCN, although SKP2 knockdown only enhanced p27 stabilisation in the Tet21N MYCN+ cells at the 24Hr time-point (Fig. 2A). A 72 hr exposure to SKP2 siRNA significantly reduced cell viability, compared to the negative control siRNA (NCS), in the presence and absence of MYCN (Fig. 2B, p < 0.01, paired t test). However, cell cycle arrest, indicated by an increase in the G1:S ratio, following 24 hr SKP2 siRNA exposure was only observed in Tet21N MYCN+ cells suggesting that p27 stabilisation at this time-point was the main
contributor to cell cycle arrest (Fig. 2A and C). Control Tet21N MYCN− cells have a markedly higher proportion of cells in G1 compared to Tet21N MYCN+ cells, which have a larger S phase population consistent with the role of MYCN in regulating cell cycle progression (Fig. 2D) [5]. The effect on the G1:S ratio in Tet21N MYCN+ cells may therefore reflect, in part, the lower baseline G1 population of 50% as opposed to the Tet21N MYCN- cells where the baseline G1 population is already very high at 80%.
Please cite this article in press as: Laura Evans, Lindi Chen, Giorgio Milazzo, Samuele Gherardi, Giovanni Perini, Elaine Willmore, David R. Newell, Deborah A. Tweddle, SKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.03.044
ARTICLE IN PRESS 4
L. Evans et al./Cancer Letters ■■ (2015) ■■–■■
Fig. 2. MYCN expression influences the cell cycle response to SKP2 siRNA knockdown but not the effects on cell viability and apoptosis. (A) Western blot analysis of SKP2, p27 and p21 following SKP2 siRNA exposure for the time-periods indicated. (B) Cell proliferation assay following 72 hr exposure to siRNA. Data are normalised to the nontransfected control (NT) and compared to the negative control siRNA (NCS) (paired t test, p ≤ 0.01). (C) G1:S ratio following 24 hr treatment with siRNA. SKP2 knockdown is compared to the negative control siRNA (NCS) (paired t test, p ≤ 0.05). (D) Cell cycle phase analysis after 24 hr siRNA treatment. (E and F) Apoptotic cell death represented by an increase in the sub-G1 DNA fraction (E) and caspase 3/7 activity (F) following exposure to SKP2 siRNA for 48 hr and 24 hr respectively and compared to the negative siRNA control (NCS), * and ** correspond to p values of
Contents lists available at ScienceDirect
Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
SKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma Laura Evans a, Lindi Chen a, Giorgio Milazzo b, Samuele Gherardi b,c, Giovanni Perini b,c, Elaine Willmore a, David R. Newell a, Deborah A. Tweddle a,* a
Newcastle Cancer Centre at the Northern Institute for Cancer Research, Newcastle University, Newcastle Upon Tyne NE2 4HH, UK Department of Pharmacy and Biotechnology, University of Bologna, Via F. Selmi 3, Bologna 40126, Italy c Health Science and Technologies-Interdepartmental Centre for Industrial Research (HST-ICIR), University of Bologna, Via Tolara di Sopra 41/E, Ozzano Emilia (Bologna) 40064, Italy b
A R T I C L E
I N F O
Article history: Received 20 January 2015 Received in revised form 26 March 2015 Accepted 28 March 2015 Keywords: SKP2 MYCN Neuroblastoma G1 arrest Apoptosis
A B S T R A C T
SKP2 is the substrate recognition subunit of the ubiquitin ligase complex which targets p27KIP1 for degradation. Induced at the G1/S transit of the cell cycle, SKP2 is frequently overexpressed in human cancers and contributes to malignancy. We previously identified SKP2 as a possible MYCN target gene and hence hypothesise that SKP2 is a potential therapeutic target in MYCN amplified disease. A positive correlation was identified between MYCN activity and SKP2 mRNA expression in Tet21N MYCN-regulatable cells and a panel of MYCN amplified and non-amplified neuroblastoma cell lines. In chromatin immunoprecipitation and reporter gene assays, MYCN bound directly to E-boxes within the SKP2 promoter and induced transcriptional activity which was decreased by the removal of MYCN and E-box mutation. Although SKP2 knockdown inhibited cell growth in both MYCN amplified and non-amplified cells, cell cycle arrest and apoptosis were induced only in non-MYCN amplified neuroblastoma cells. In conclusion these data identify SKP2 as a direct transcriptional target of MYCN and supports SKP2 as a potential therapeutic target in neuroblastoma. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction MYCN amplification is found in 25% of neuroblastoma and is strongly associated with advanced-stage and aggressive disease [1,2]. Although different strategies for inhibiting MYCN have been investigated, direct therapeutic targeting of the oncoprotein presents a significant challenge [3,4]. We have previously identified a number of genes involved in the regulation of the G1 checkpoint whose expression changes in relation to MYCN status [5]. One potential MYCN target gene is the F-box protein S-phase kinase-associated protein 2 (SKP2) which is the substrate recognition subunit for the SCFSKP2 E3 ligase complex. Implicated primarily in G1/S progression, SKP2 targets cell cycle regulators (e.g. p27KIP1, p21CIP1 and p57KIP2) for ubiquitination and degradation, thereby promoting cell cycle progression. Overexpressed in multiple cancer types, the main oncogenic contribution of SKP2 is attributed to targeting the CDK inhibitor
Abbreviations and acronyms: SKP2, S-phase kinase-associated protein 2; SCF, SkpCullin-F-box containing complex; ChIP, chromatin immunoprecipitation; CDK, cyclin dependent kinase; MNA, MYCN amplified; NCS, negative control siRNA. * Corresponding author. Tel.: +44 0 191 208 4421; fax: +44 (0) 191 208 4301. E-mail address: [email protected] (D.A. Tweddle).
p27KIP1 (p27 hereafter) for degradation, with the inverse relationship of high SKP2 and low p27 level often correlating with poor patient prognosis [6–8]. SKP2 overexpression has been identified as an indicator of highrisk neuroblastoma and found to correlate with low p27 protein levels and high E2F activity, suggesting SKP2 contributes to disease development and progression [9]. Moreover, RNA studies have previously demonstrated a relationship between MYCN and SKP2 expression in primary neuroblastoma tumours and cell lines [5,9], suggesting SKP2 may be a potential MYCN target gene. SKP2 has recently been identified as a direct c-MYC target gene [10]. Due to the homology within the Myc family and functional overlap often observed between c-MYC and MYCN, established c-MYC targets are often automatically presumed to be regulated by MYCN. However, different expression patterns are observed for these transcription factors in normal development and as oncogenes they are involved in distinct tumour types (e.g. c-MYC in Burkitt’s lymphoma and MYCN in neuroblastoma). Furthermore not all c-MYC gene targets are found to be expressed in MYCN-expressing neuroblastoma cells most probably due to the reported differences in E-box selection between MYCN and c-MYC [11], as reviewed in Refs. 12 and 13. We therefore hypothesise that SKP2 is a direct MYCN target gene and contributes to the aggressive phenotype of MYCNamplified tumours.
http://dx.doi.org/10.1016/j.canlet.2015.03.044 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Laura Evans, Lindi Chen, Giorgio Milazzo, Samuele Gherardi, Giovanni Perini, Elaine Willmore, David R. Newell, Deborah A. Tweddle, SKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.03.044
ARTICLE IN PRESS L. Evans et al./Cancer Letters ■■ (2015) ■■–■■
2
To investigate this hypothesis, we assessed the correlation between MYCN and SKP2 protein, and SKP2 mRNA expression in the conditional MYCN-expressing SHEP Tet21N cells and a panel of MYCN amplified and non-amplified neuroblastoma cell lines. Using chromatin immunoprecipitation (ChIP) and reporter gene assays, this is the first study to show that SKP2 is a direct MYCN transcriptional target. In addition, using SKP2 siRNA knockdown we highlight the therapeutic potential of targeting SKP2 in neuroblastoma.
Chromatin immunoprecipitation (ChIP assay) The ChIP assay was performed as previously described [22], and the antibodies employed were: MYCN (B8.4.B, C-22, Santa Cruz,), c-MYC (N-262, Santa Cruz) and MAX (C-17, Santa Cruz). Samples were analysed using SYBR Green quantitative PCR with the specific primers listed in the Supplementary material (Table S1). The known MYCN target gene ornithine decarboxylase (ODC1) was used as a positive control using forward primer 5′- ATCACTTCCAGGTCCCTTGC-3′ and reverse primer 5′-GAGAGCGGAAAAGGGAAATC-3′. Luciferase assay
Materials and methods Cell culture and reagents MYCN-amplified neuroblastoma cell lines used were LAN5 [14], IMR32 [15] and p53 mutant IMR-KAT100 [16] and SK-N-BE(2c) [17] cells. Non-MYCN amplified cell lines used were SHSY5Y [17], GIMEN [18] and p53 mutant SKNAS [19]. Tet21N cells were cultured in media containing 1 μg/ml tetracycline (Sigma-Aldrich) for 24 hrs prior to experiments to switch off MYCN expression [20]. The identity of all cell lines was confirmed by karyotyping and was consistent with published reports. Cells were cultured in RPMI medium (Sigma) with 10% (v/v) foetal bovine serum (FBS). A total of 200 μg/ml of G418 antibiotic (Sigma-Aldrich) was added to the Tet21N cell medium. All cell lines were tested regularly and found to be free from Mycoplasma.
Quantitative reverse transcription PCR (qRT-PCR) qRT-PCR was performed as previously described and analysed using the ΔCT/ ΔΔCT method [5]. All primers and probes were inventoried using TaqMan Gene Expression Assays (Applied Biosystems) and mRNA expression data normalised to GAPDH.
The SKP2 reporter construct -1148-SKP2 and pGL3basic vector were kindly donated by Prof. Javier Lèon (University of Cantabria, Spain) [10]. pGL3-MutEB was generated by site-directed mutagenesis replacing identified E-Boxes (CACCTG) and (CCCGTG) with CTGCAG, and confirmed by sequencing. Cells were transfected with 0.8 μg of reporter constructs and 0.1 μg of β-galactosidase (pCMV.SPORT.βgal, Invitrogen) using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured using the luciferase assay system from Promega (E4030), corrected for nontransfected controls and normalised using β-gal activity to control for transfection efficiency. Statistical analyses All statistical tests were two sided and performed using GraphPad Prism (Graphpad Software, Inc.). The level of significance was taken to be p ≤ 0.05.
Results SKP2 protein expression correlates with MYCN expression in MYCNregulatable Tet21N cells
Western analysis Western blotting was performed using previously described methods [21]. Primary antibodies used were SKP2 (32–3300, Invitrogen) at 1:1000, MYCN (SC-53993, SantaCruz) at 1:500, p27 (SC-528, Santa-Cruz) and p21 (OP64, Calbiochem) both at 1:100 and GAPDH (SC-25778, Santa-Cruz) at 1:1000. Secondary goat anti-mouse/rabbit horseradish peroxide-conjugated antibodies (P0447/P0448, Dako) were used at 1:1000.
Cycloheximide treatment Tet21N cells were treated with final concentrations of tetracycline (1 μg/ml) and/ or 25 μmol/l cycloheximide (Calbiochem), alone or in combination. Cells were harvested at the indicated times and analysed using Western blotting.
RNA interference siRNA (40 nM) was transfected into cells using Lipofectamine 2000 reagent (Invitrogen) which was replaced with RPMI medium containing 10% (v/v) FBS after 24 hr. Three different oligonucleotides targeting SKP2 were synthesised by Eurogentec and the most effective duplex used in all knockdown experiments. siRNA targeting CDKN1B (p27) was verified and purchased from Qiagen (SI02621990). The sense strand of the SKP2 siRNA sequence chosen was: GUGAUAGUGUCAUGCUAAA. Inhibition of expression was confirmed by Western blotting against a negative control siRNA (NCS).
Cell proliferation assay Cell viability was measured by the sodium 3′-{1-(phenylaminocarbonyl)-3,4tetrazolim]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) colorimetric assay (Roche Applied Science) according to the manufacturer’s instructions.
Flow cytometry Cells were harvested, washed in cold PBS, fixed in 70% (v/v) ethanol and stored at 4 °C. Fixed cells were rinsed twice with PBS and treated with propidium iodide (PI) solution containing 40 μg/ml PI (Sigma) and 0.1 mg/ml RNase A (Sigma) at 37 °C for 30 minutes. Stained cells were analysed by a FACSCalibur cytometer (BD Bioscience, Oxford, UK).
Tet21N MYCN- cells showed an ~3-fold reduction in SKP2 mRNA 24 hr after tetracycline addition (Fig. 1A), which corresponded with decreased SKP2 protein expression over the 5 day tetracycline exposure period (Fig. 1B). Reduced SKP2 expression was associated with increased protein levels of the SKP2 targets p27 and p21CIP1 (p21 hereafter), suggesting that stabilisation of these CDK inhibitors induced the growth inhibition observed in the exponentially proliferating Tet21N MYCN− cells (Fig. 1C). To confirm that stabilisation of the CDK inhibitors was a result of decreased SKP2 protein expression, Tet21N MYCN+ cells were treated with a combination of tetracycline and the mRNA translation inhibitor cycloheximide (25 μmol/l, Fig. 1D). Cycloheximide treatment decreased MYCN and SKP2 protein expression (lanes 3 and 4), mirroring the effect of tetracycline only (lanes 5–7), and reduced the level of p27 and p21. Only p27 protein was still present, although at a reduced level, after combination treatment (lanes 8 and 9) suggesting that the loss of SKP2 in Tet21N MYCN− cells resulted in the post-translational stabilisation of p27, but not p21. Data analysis from the Versteeg-88 dataset of clinical neuroblastoma tumour samples by the R2 microarray analysis and visualisation platform (http://r2.amc.nl) [23] showed that expression of SKP2 and MYCN were strongly correlated (Supplementary material, Fig. S1 [23]) which was confirmed in two additional data sets; Maris-101, r = 0.520, p = 2.6e−8 and Jagannathan-100, r = 0.528, p = 1.7e−8 (figures not shown, http://r2.amc.nl). To further investigate the relationship between MYCN and SKP2, mRNA and protein levels were analysed in a panel of MYCN amplified (MNA) and nonMYCN amplified (non-MNA) neuroblastoma cell lines. MYCN protein levels correlated with SKP2 mRNA levels (Pearson’s correlation, r2 = 0.62, p < 0.05, Fig. 1E) but not SKP2 protein expression (Fig. 1F) suggesting that MYCN may influence the numerous mechanisms reported to regulate SKP2 protein stability.
Caspase-3/7 assay After 24 hr siRNA exposure a 1:1 volume of Caspase-Glo 3/7 reagent (Promega) was added to each well and incubated in the dark for 1 hr at room temperature. The plate was analysed on a microplate luminometer (Berthold Technologies, Hertfordshire, UK).
MYCN sensitises Tet21N cells to SKP2 knockdown SKP2 protein was successfully knocked down using siRNA (Fig. 2a), and resulted in a clear increase in p21 protein levels independent
Please cite this article in press as: Laura Evans, Lindi Chen, Giorgio Milazzo, Samuele Gherardi, Giovanni Perini, Elaine Willmore, David R. Newell, Deborah A. Tweddle, SKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.03.044
ARTICLE IN PRESS L. Evans et al./Cancer Letters ■■ (2015) ■■–■■
3
Fig. 1. SKP2 expression is associated with MYCN expression in neuroblastoma cell lines. (A) Normalised expression of SKP2 mRNA levels in Tet21N MYCN+ and MYCN− cells following 24 hr exposure to tetracycline (paired t test, p ≤ 0.05). (B) Western blot analysis of protein expression of SKP2, p27 and p21 in Tet21N cells in the presence and absence of tetracycline (C) Tet21N MYCN+ cells proliferate more rapidly than Tet21N MYCN− cells. (D) Western blot analysis of MYCN, SKP2, p27 and p21 protein levels in Tet21N cells following treatment with tetracycline (TET) and cycloheximide (CHX). MYCN protein acted as positive control due to its short half-life (~2 hr). (E and F) Relationship of MYCN and SKP2 at transcript (E) and protein (F) level in a panel of neuroblastoma cell lines (Spearman correlation, r = 0.62, p ≤ 0.05).
of MYCN, although SKP2 knockdown only enhanced p27 stabilisation in the Tet21N MYCN+ cells at the 24Hr time-point (Fig. 2A). A 72 hr exposure to SKP2 siRNA significantly reduced cell viability, compared to the negative control siRNA (NCS), in the presence and absence of MYCN (Fig. 2B, p < 0.01, paired t test). However, cell cycle arrest, indicated by an increase in the G1:S ratio, following 24 hr SKP2 siRNA exposure was only observed in Tet21N MYCN+ cells suggesting that p27 stabilisation at this time-point was the main
contributor to cell cycle arrest (Fig. 2A and C). Control Tet21N MYCN− cells have a markedly higher proportion of cells in G1 compared to Tet21N MYCN+ cells, which have a larger S phase population consistent with the role of MYCN in regulating cell cycle progression (Fig. 2D) [5]. The effect on the G1:S ratio in Tet21N MYCN+ cells may therefore reflect, in part, the lower baseline G1 population of 50% as opposed to the Tet21N MYCN- cells where the baseline G1 population is already very high at 80%.
Please cite this article in press as: Laura Evans, Lindi Chen, Giorgio Milazzo, Samuele Gherardi, Giovanni Perini, Elaine Willmore, David R. Newell, Deborah A. Tweddle, SKP2 is a direct transcriptional target of MYCN and a potential therapeutic target in neuroblastoma, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.03.044
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Fig. 2. MYCN expression influences the cell cycle response to SKP2 siRNA knockdown but not the effects on cell viability and apoptosis. (A) Western blot analysis of SKP2, p27 and p21 following SKP2 siRNA exposure for the time-periods indicated. (B) Cell proliferation assay following 72 hr exposure to siRNA. Data are normalised to the nontransfected control (NT) and compared to the negative control siRNA (NCS) (paired t test, p ≤ 0.01). (C) G1:S ratio following 24 hr treatment with siRNA. SKP2 knockdown is compared to the negative control siRNA (NCS) (paired t test, p ≤ 0.05). (D) Cell cycle phase analysis after 24 hr siRNA treatment. (E and F) Apoptotic cell death represented by an increase in the sub-G1 DNA fraction (E) and caspase 3/7 activity (F) following exposure to SKP2 siRNA for 48 hr and 24 hr respectively and compared to the negative siRNA control (NCS), * and ** correspond to p values of
